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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
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Indole-3-carbinol and 3’, 3’-diindolylmethane modulate androgen's effect on C-C
chemokine ligand 2 and monocyte attraction to prostate cancer cells
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Eun-Kyung Kim, 2 Young S. Kim, 2+John A. Milner and 3Thomas T.Y.Wang
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Nutritional Sciences Research Group, Division of Cancer Prevention, National Cancer Institute, NIH,
Bethesda, MD, USA
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Department of Nutrition and Food Science, University of Maryland, College Park, MD, USA
Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural
Research Service, US Department of Agriculture, Beltsville, MD, USA
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Running Title: I3C and DIM inhibit monocyte attraction.
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Key words: prostate cancer, broccoli, monocyte, migration, CCL2,
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This work was supported by U.S appropriated funds to USDA project number 1235-51530-053-00D
(TTYW) and the National Cancer Institute (YSKE-K K, JAM).
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Corresponding Author: Thomas T.Y. Wang, Phone: 304-504-8459; Fax: 304-504-9062; E-mail:
tom.wang@ars.usda.gov.
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# Current address: Division of Food Bioscience, Department of Natural Science, Konkuk University,
Chungju 380-701, Korea
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+ Current address: Beltsville Human Nutrition Research Center, Agricultural Research Service, US
Department of Agriculture, Beltsville, MD, USA
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No conflict of interest.
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Word count: Text 3054, 6 Figures
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Abstract
Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor
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site is mediated by C-C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We
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hypothesized that androgen could modulate CCL2 expression in hormone-responsive prostate cancer cells
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and thereby promote recruitment of monocytes. Given the inhibitory effect of broccoli-derived compounds
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indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on androgen-dependent pathways, we also
32
reasoned that I3C and DIM could modulate the effect of androgen on CCL2-mediated pathways.
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Dihydrotestosterone (DHT) was found to induce a time (0-72 hrs) and concentration-dependent (0-1 nM)
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increase in CCL2 mRNA levels in androgen-responsive human prostate cancer cells (LNCaP). This
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increase in CCL2 mRNA corresponded with increased secretion of CCL2 protein. The effect of DHT was
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mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA against AR
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negated the induction of CCL2. Although DHT also induced TWIST1 mRNA, an epithelial-mesenchymal
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transition related factor and purported inducer of CCL2, blocking its expression with small inhibitor RNA
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did not inhibit DHT induction of CCL2 mRNA. Moreover, conditioned media from androgen-treated cells
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promoted human monocyte THP-1 cell migration and this effect was blocked by antibody against CCL-2.
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Both I3C and DIM inhibited promotional effects of DHT on CCL2 and migration. These results
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demonstrate that androgen may regulates CCL2 and promotes inflammatory micro-environments in
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prostate tumors, and that this process can be blocked by broccoli-derived compounds.
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Introduction
Androgens are functionally required for the normal growth and development of the prostate gland. In
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adult males, androgens promote secretary epithelial cell survival. However, androgens also promote
48
prostate tumor development and progression (1,2). Androgen deprivation is the only clinically effective
49
therapy for advanced prostate cancer. However, because of the relapse of castration-resistant androgen
50
independent tumors, the long-term benefit of androgen deprivation in patients with metastatic disease
51
remains controversial (3,4). Although previous research shows many of androgens biological effect are
52
likely through regulation of androgen responsive genes (ARG) via an androgen receptor-mediated
53
pathway (5), some of the molecular effects of androgens in normal and prostate cancer remain unresolved.
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Inflammation as a causal agent has been linked to approximately 20% of human cancers (6). In prostate
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cancer, a growing amount of evidence suggests a link between prostate inflammation and subsequent
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cancer development (7-12). Previous studies suggest C-C chemokine ligand 2 (CCL2), also known as
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monocyte chemoattractant protein-1 (MCP-1) may play pivotal role in prostate cancer tumorigenesis and
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invasion (13,14). CCL2 is known to attract monocytes to the site of inflammation, and by binding to its
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receptor CCR2, directly stimulates prostate cancer cell proliferation, survival, and migration (15). Prostate
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cancer cells LNCaP, C4-2B, PC-3, and VCaP produce CCL2 (16,17). Furthermore, recent findings suggest
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a role for CCL2 in acquisition of epithelial-mesenchymal transition (EMT) properties (18). EMT has been
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shown to be crucial for the pathogenesis of tissue fibrosis and cancer (19). Prostate tumor epithelial cells
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gain the ability to migrate and invade by differentiating through activation of biological pathways
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associated with EMT. Androgens were reported to induce changes that are characteristic of EMT such as
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expression of TWIST-1, CDH1 and SNAIL, cytoskeleton reorganization, and promotion of metastatic
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behavior of castration-resistant prostate cancer cells (20). TWIST-1, a helix-loop-helix transcription factor
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thought to be an important mediator of EMT, is up-regulated in several types of epithelial cancers
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including prostate, breast and gastric carcinomas (21-24). Several EMT-related genes such as CDH1,
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CDH2, SNAIL, desmin, vimentin, are putative down-stream targets regulated by TWIST-1. TWIST-1 also
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appears to be involved in inflammatory pathways (25). TWIST-1 expression in T helper 1 (Th1)
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lymphocytes and bone marrow-derived macrophages attenuates the expression of interferon-γ, IL-2, and
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TNF-α, further implicating TWIST-1 in the regulation of cytokine expression (25-27). It was also reported
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that CCL2 expression may be subject to regulation by TWIST-1 (28). The relationship between androgens
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and these EMT-related proteins, as well as the relation between TWIST-1 and CCL2 in prostate cells
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remain unresolved.
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Cancer prevention represents an integral part of a sound strategy in fight against cancer. Diet-derived
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compounds are often the focus due to their perceived safety, Indole-3-carbinol (I3C) is a putative
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preventive compound derived from hydrolysis of glucobrassicin from ingesting cruciferous vegetables
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such as broccoli, cabbage and cauliflower (29). I3C can be converted to the dimeric form 3’,3’-
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diindolylmethane (DIM) in stomach (30,31). Previous studies, including our own, have shown that the I3C
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and DIM inhibit prostate cancer cell growth in vitro and in vivo (32-34). Both I3C and DIM appeared to
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exert their effects in part through modulation of androgen-dependent pathways. In addition, these agents
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have also been reported to prevent or delay the progression of cancer, through their ability to attack cancer
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stem cells or EMT-type cells and modulate inflammation in cancer cells (35,36). The effects of these
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compounds on prostate cancer-related EMT remains unknown.
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Given the potential roles of androgen in regulating prostate cancer and EMT and existent information on
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TWIST-1 and CCL-2, we hypothesized that androgen may modulates TWIST-1 and in turn CCL2
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expression in prostate cancer cells, and these effect may resulted in promotion of monocytes recruitment.
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The present study seeks to resolve these interactions. Finally, given the effects of I3C and DIM on
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androgen-dependent pathways (32-34), we also sought to determine the effects of I3C and DIM on
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TWIST-1, CCL2 and monocyte migration.
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Materials and Methods
Chemicals and Reagents. I3C, DIM, dihydrotestosterone (DHT), and DMSO were from Sigma
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Chemical (St. Louis, MO). Trizol, AffinityScript Multiple Temperature cDNA Synthesis kit and Taqman
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real-time PCR primers and probes were obtained from Life Technology (Grand Island, NY). On-Target
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plus SMART pool siRNA targeting androgen receptor (AR) and TWIST-1 were purchased from Thermo
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Fisher Scientific (Waltham, MA). HiPerFect Transfection Reagent was purchased from Qiagen (Valencia,
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CA, USA).
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Cell culture. LNCaP and PC-3 human prostate cancer cells were obtained from the American Type
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Culture Collection (Manassas, VA, USA) and maintained in Media A (RPMI 1640 medium with phenol
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red and 2mM L-glutamine (Invitrogen/Life Technologies, Grand Island, NY), 100 U/mL penicillin and
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100 mg/mL streptomycin (BioSource International, Camarillo, CA, USA) with 10% fetal bovine serum
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(FBS) (Invitrogen/Life Technologies). For androgen-related experiments, cells were seeded as follows: 6-
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well plates at 250,000 cells/well. After 24 h in medium A, cells were switched to medium B, which had
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the same composition as Media A except that FBS was replaced with 10% charcoal dextran-treated FBS
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(GIBCO/Life Technologies) for an additional 24 h to minimize background androgen levels. Cells were
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incubated in the presence of 5% CO2 in air at 37°C. THP-1 cell were also purchased from American Type
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Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 medium with phenol red and
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2mM L-glutamine (Invitrogen/Life Technologies, Grand Island, NY), 100 U/mL penicillin and 100
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mg/mL streptomycin (BioSource International, Camarillo, CA, USA) with 10% fetal bovine serum (FBS)
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(Invitrogen/Life Technologies)
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RNA Isolation and Real-Time Reverse Transcriptase PCR. Total RNA was isolated using TRIzol
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reagent (Invitrogen/Life Technologies, Grand Island, NY), and reverse transcribed to cDNA using
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AffinityScript Multiple Temperature cDNA Synthesis kit (Agilent/Life Technologies). Real-time PCR was
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carried out using a TaqMan Fast Universal PCR Master Mix on a 7900HT FAST real-time PCR System
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(Applied Biosystems/Life Technologies). The amplification parameters used were as follows: 95°C for 20
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s, followed by 46 cycles of amplification at 95°C for 1 s and 60°C for 20 s. Relative mRNA fold changes
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to control were calculated using the comparative CT (2−ΔΔCt) cycle (ΔCt) method following
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manufacturer’s directions. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an
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endogenous control for all gene expression analysis calculation.
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Determination of gene expression in LNCaP cells using RT-PCR. To examine the effects of test
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compounds on TWIST1, CDH1, CDH2, SNAI1 (SNAIL1), DES (desmin), VIM (vimentin), KLK3 (PSA),
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CCL2 or TNF (TNF-α) mRNA levels, LNCaP cells were plated in six-well plates (0.25×106 cells/well) in
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Media A and after 24 h the media was removed and replaced with fresh media containing vehicle or varied
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concentrations of test compounds I3C at 5, 25, 50 µM and DIM at 1, 5, 10 µM. LNCaP cells were plated
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in six-well plates (0.25×106 cells/well) in Media A, and switched to Media B 24 h after plating to
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minimize the effect of serum hormones. The cells were then incubated in Media B for an additional 24 h
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before the treatments began. Twenty-four hours later, the medium was replaced with fresh medium
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containing 1 nM DHT with or without test compounds. For all experiments, fresh medium containing the
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test compounds was changed daily and cells were harvested for total RNA isolation using the Trizol
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method (Invitrogen/Life Technologies) after 48 h. Taqman real-time PCR was used to quantify expression
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of the mRNA..
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Small interfering RNA (siRNA) oligonucleotides studies. Small interfering RNA specifically block
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expression of AR or TWIST1 mRNA was used to confirm the role of androgen regulation of TWIST1,
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CCL2 and TNF-α mRNA expression. LNCaP cells were seeded in 6-well plates as described above.
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Twenty-four hrs after androgen deprivation, LNCaP cells were treated with or without 1 nM
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dihydrotestosterone (DHT). For siRNA experiments, cells were transfected with 5 nM of small interfering
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RNA oligonucleotides targeting AR or TWIST1 using HiPerFect transfection reagent according to the
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manufacturer's protocol. Forty-eight hours after transfection, cell culture media were replaced with fresh
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media in the presence or absence of 1 nM DHT for an additional 48 h. After treatment, cells were
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harvested for total RNA isolation as described above.
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Trans-well cell migration assay. For trans-well migration assays, 2 × 105 THP-1 cells in 0.2 ml phenolred free RPMI1640 (Invitrogen/Life Technologies) were placed into the non-coated membrane top
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chamber (24-well insert; pore size, 8 μm; Corning Costar) and allowed to migrate toward LNCaP cells-
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conditioned media (1.2 ml) in the lower chamber. After 10 hours of incubation with the conditioned media,
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cells migrated to the lower chamber were collected by centrifugation (1000 g x 10 min), re-suspend in 0.1
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ml PBS and the number of cells counted under a light microscope (Olympus CK40) using a
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hemocytometer. To confirmed that CCL2 in conditioned media contributed toward migration of THP-1
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cells, condition media were first treated with 0.5 μg/ml anti-human neutralizing CCL2 antibody (R&D
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Systems) or IgG isotype control (BD Pharmingen) for 1hr , then migration assays were conducted as
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above.
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Enzyme-linked immunosorbent assay (ELISA) determination of CCL2 and TNF-α protein. LNCaP
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cell were treated with or without DHT (1 nM) in presence or absence of DIM (10 μM) or I3C (50 μM) for
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48 h. After incubation, LNCaP cells conditioned media was removed, lyophilized and re-suspended in
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RPMI-1640 phenol red free media (Invitrogen/Life Technologies) to yield 200 µg protein/µL, 50 μl was
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used for detection of CCL2 or TNF-α. Protein expressions of CCL2 and TNF-α were determined by using
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commercially available ELISA kits. EH2MCP1 for CCL-2, EHTNFA for TNF-α (Thermo Scientific,
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Rockford, IL, USA) according to the manufacturer's instructions. The optical density (OD) was
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determined at 550 nm or 450 nm using a multi-well plate reader (Spectra Max Plus, Molecular Devices,
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Sunnyvale, CA, USA).
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Statistical methods. Statistical analysis of data was carried out with the GraphPad PRISM 4 program
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(GraphPad Software, Inc., La Jolla, CA). Multiple group data were analyzed using one-way or two-way
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ANOVA followed by a Bonferroni post-hoc test. An unpaired Student's t test was used to compare
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experiments between two groups. Gene expression results are expressed as means ± SEM relative to
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vehicle control. Data are representative of three independent experiments.
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Results
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Androgen Induces TWIST1 mRNA Expression through an AR-dependent pathway. DHT (0-1nM)
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significantly induced TWIST1 in a dose and time-dependent manner (Fig. 1A and B). As shown in Fig.
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1C and D, adding the AR siRNA effectively inhibited DHT induction of the well-documented androgen
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responsive gene PSA (Fig. 1C). Similarly, AR siRNA also inhibited DHT induction of TWIST-1 mRNA
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(1D).
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Androgen differentially Regulate putative TWIST-1 Down-Stream Target genes mRNA
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Expression. DHT- treatment of LNCaP cells lead to differential effects on TWIST-1-responsive gene
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mRNA expression. EMT-related, TWIST-1-responsive genes such as, CDH1, SNAIL and VIM were not
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affected by DHT treatment (Fig. 2A, B and C), and the expression of DES and CDH2 was below detection
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limits under the current experimental condition. By contrast, CCL2 and TNF, two inflammation-related
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putative TWIST-1-responsive genes, were induced by DHT treatment in a time- and dose-dependent
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manner (Fig. 2D to G).
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Androgen Regulation of CCL2 and TNF-α mRNA is through an AR but not a TWIST-1 dependent
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pathway. Both androgen receptor and TWIST1 may both contribute to the up-regulation of CCL2 and
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TNF mRNA levels by DHT-treatment. Small interfering RNA against AR effectively inhibited DHT
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(1nM) induced increase in CCL2 and TNF-α mRNA levels (Fig. 3A, B). In the contrast, siRNA against
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TWIST1 did not affect the expressions of DHT-induction of CCL2 or TNF (Figure 3 C, D). Tretament of
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cells with siRNA for AR and TWIST1 lead to ~90% and ~ 80% inhibition of AR and TWIST expression,
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respectively (Figure 3E). TWIST1, CCL2 and TNF-α mRNA expression levels were higher in PC-3 cells
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than in LNCaP cells (Fig. 4A), Furthermore, treatment of PC-3 cells with DHT (1 nM) did not affect the
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mRNA expression levels of these genes (Fig. 4B, C). Consistent with increase in CCL2 mRNA levels,
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DHT also lead to an increase in CCL2 protein levels (Fig. 5A)
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Conditioned Media from DHT-treated LNCaP cell Increase THP-1 Monocyte Migration. As
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shown in Fig. 5B, the number of THP-1 cells migrated toward DHT-treated LNCaP cells was significantly
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higher than in those provided the control media. Adding DHT (1 nM) directly into media did not lead to
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increase in migration of THP-1 cells. Pre-treatment of conditioned media with a neutralizing antibody
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against CCL2 was found to attenuate increased migration of THP-1 cells toward DHT-treated conditioned
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media (Fig. 5C).
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I3C and DIM Modulate Effects of Androgen on CCL2 and Migration. Consistent with their effect on
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androgen-dependent pathways, I3C and DIM exerted a concentration-dependent inhibition of both
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TWIST1 and CCL2 mRNA expression induced by DHT treatments. This response was similar to the
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effects of I3C and DIM had on androgen induction of the well-documented androgen responsive gene PSA
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mRNA (Fig. 6A, B and C). Furthermore, treatment of cells with I3C (50 μM) and DIM (10 μM) also
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inhibited the DHT-induced increase in CCL2 protein (Fig. 6D and E). Furthermore, I3C (50 μM) or DIM
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(10 μM) treatment also led to significantly reduction of DHT-induced migration of THP-1 cells (Fig. 6F
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and G).
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Discussion
The molecular mechanisms underlying prostate cancer’s etiology and progression remain unclear.
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Although exposure to androgen is consider one of the risk factor for prostate cancer, the mechanisms also
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not completely delineated. In the present study we reported a novel finding that CCL2, a protein critical in
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attracting monocytes (37,38), is an androgen-responsive gene. We show that exposure of the androgen-
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responsive LNCaP cell line to DHT led to induction of CCL2 mRNA as well as protein (Figure 5A) and
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this effect of DHT on CCL2 mRNA was blocked by siRNA against AR (Figure 3A). This observation
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supports the notion that exposure to androgens can promote attraction of monocytes by prostate cancer
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cells. We provide in-vitro data (Figure 5C) to support this notion as we observed increased migration of
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THP-1 toward conditioned media derived from DHT-treated cells (Fig. 5B). The involvement of CCL2
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was further strengthened, because THP-1 cell migration was inhibited when conditioned media was pre-
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incubated with anti-CCL-2 antibody (Fig. 5C). These results suggest that exposure of prostate cancer cells
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to androgens may create a pro-inflammatory environment, where the prostate cancer cell is stimulated to
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secrete CCL2 and attract monocytes toward tumor site. Monocytes can produce cytokines such as IL-6
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and IL-1β (39). These cytokines are known to stimulate prostate cancer cell growth and angiogenesis (40).
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Therefore, up-regulation of CCL2 may be a mechanism whereby androgens promotes prostate cancer
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development. We also observed that the androgen-independent prostate cancer cell PC-3 express higher
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levels of CCL2 mRNA. PC-3 cell is considered to be more advance than LNCaP cell in prostate cancer
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tumorigenesis process. The higher expression of CCL2 in PC-3 cells as compared to LNCaP cells is
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consistent with PC-3’s more aggressive phenotype, and suggest that they may have a grater capacity for
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monocyte recruitment. This will lead to increased inflammation in the tumor site. One may thus reason
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that anti-inflammatory agents targeting the CCL2 pathway may delay tumor growth in both androgen-
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dependent and -independent prostate tumors.
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One of our initial hypotheses was that DHT could modulate EMT. Epithelial-mesenchymal transition
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induction in cancer cells results in the acquisition of invasive and metastatic properties. Epithelial-
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mesenchymal transition -type cells, which share molecular characteristics with cancer stem cells (CSCs),
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are believed to play critical roles in early cancer metastasis and drug resistance as demonstrated in several
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human malignancies including prostate cancer (41-43). We found that LNCaP cells exposed to DHT
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significantly up-regulated the EMT-related protein TWIST1 mRNA levels. This effect of DHT on
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TWIST1 is dependent on AR as siRNA against AR blocked the induction of TWIST1 mRNA by DHT
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(Fig. 1). TWIST1, a helix-loop-helix transcription factor, is highly expressed in many types of human
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cancers (44). Recently, TWIST-1 was suggested to be an oncogene (45-47) and confers prostate cancer
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cells with an enhanced metastatic potential through promoting EMT and a high TWIST1 expression in
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human prostate cancer is associated with an increased metastatic potential (48). However, we did not find
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DHT to effect the TWIST-1-regulated and/or EMT-related genes such as CDH1, SNAIL and vimentin
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expression in LNCaP cells (Fig. 2A-C). These results suggest that although androgens induced TWIST-1,
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they did not lead to development of an EMT phenotype in our experimental conditions. TWIST1 was also
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reported to be a regulator of CCL2 (49). We did found DHT treatment increased the mRNA of two
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purported TWIST-1 responsive genes, CCL2 and TNF-α,. TNF-α is also a potent chemoattractant for
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several cell lines (50). However, the effect of androgen on CCL2 and TNF-α mRNA appeared to be AR-
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dependent pathway but not TWIST1 dependent. Androgen receptor siRNA and not TWIST1 siRNA
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blocked the effects of DHT on CCL2 and TNF- α mRNA. These results suggest that TWIST1 is not
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involved in the regulation of CCL2 or TNF-α mRNA by DHT in LNCaP cells. Additional studies are
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necessary to elucidate the role of DHT in EMT as well as TWIST1 in the prostate cancer/EMT
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relationship.
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We previously reported I3C and DIM can inhibit LNCaP cell growth that correlated with inhibition of
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androgen-dependent pathways (33). Consistent with this observation, cells treated with I3C and DIM also
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significantly blocked the induction of CCL2 mRNA by DHT (Fig. 6C). In addition, I3C and DIM
252
treatment also led to the inhibition of CCL2 protein expression induced by DHT as well as migration of
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THP-1 monocytes induced by DHT (Fig. 6F,G). These results indicate that exposure of cells to I3C and
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DIM not only modulates the growth of androgen responsive prostate cancer cells but also indirectly
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influences luminal cells such as the monocytes. We reason that I3C and DIM, through inhibition of
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monocyte to the tumor site, could minimize the exposure of tumors to cytokines such as IL-1β and IL-6.
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This effect may thus attenuate the inflammatory process and ultimately lead to delay in prostate tumor
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growth and angiogenesis. Our data also support the possibility that diet or diet-derived compounds may
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act through multiple mechanisms including inflammation and angiogenesis to prevention prostate cancer.
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Additional in-vivo studies are warranted to validate our hypothesis. TWIST1, an androgen responsive gene,
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was also modulated by I3C and DIM (Figure 6B). The consequence of TWIST1 modulation by I3C and
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DIM remain unclear and warrant further study.
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In summary, we found that the androgen DHT promotes monocyte migration through up-regulation of
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CCL2 mRNA and protein. The effect of DHT on CCL2 appeared to be independent of TWIST-1 and
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mediated through AR-dependent pathways. More importantly the diet-derived factors I3C and DIM can
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modulate CCL2 and monocyte migration induced by DHT. These may be important mechanisms whereby
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diet-derived compound delay prostate tumor growth and development. Finally, we found that DHT
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induced EMT-related TWIST-1 expression but had no effect on other EMT-related genes downstream of
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TWIST-1.
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References
273
1. Meng-Lei Zhu, Natasha Kyprianou. Role of androgens and the androgen receptor in epithelial-
274
mesenchymal transition and invasion of prostate cancer cells. FASEB 2010;24:769-77.
275
2. De Marzo AM, Nelson WG, Meeker AK, Coffey DS. Stem cell features of benign and malignant
276
prostate epithelial cells. J Urol 1998;160:2381-92.
277
3. Shahinian VB, Kuo YF, Freeman JL, Goodwin JS. Risk of fracture after androgen deprivation for
278
prostate cancer. N Engl J Med 2005;352:154-64.
279
4. Makhsida N, Shah J, Yan G, Fisch H, Shabsigh R. Hypogonadism and metabolic syndrome:
280
implications for testosterone therapy. J Urol 2005;174:827-34.
281
5. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS. Androgen receptor
282
defects: historical, clinical, and molecular perspectives. Endocr Rev 1995;16:271-321.
283
6. Kuper H, Adami HO, Trichopoulos D. Infections as a major preventable cause of human cancer. J
284
Intern Med 2000;248:171-83.
285
7. De Marzo AM, Platz EA, Sutcliffe S, Xu J, Gronberg H, Drake CG, et al. Inflammation in prostate
286
carcinogenesis. Nat Rev Cancer 2007;7:256-69.
287
8. MacLennan GT, Eisenberg R, Fleshman RL, Taylor JM, Fu P, Resnick MI, et al. The influence of
288
chronic inflammation in prostatic carcinogenesis: a 5-year followup study. J Urol 2006;176:1012-16.
289
9. Mahmud S, Franco E, Aprikian A. Prostate cancer and use of nonsteroidal anti-inflammatory drugs:
290
systematic review and meta-analysis. Br J Cancer 2004;90:93-9.
291
10. Dennis LK, Lynch CF, and Torner JC. Epidemiologic association between prostatitis and prostate
292
cancer. Urology 2002;60:78-83.
293
11. Putzi MJ and De Marzo AM. Morphologic transitions between proliferative inflammatory atrophy and
294
high-grade prostatic intraepithelial neoplasia. Urology 2000;56:828-32.
295
12. Andreas B, David E, Nicolas K, Eva L, Marc SS, Johannes AS. Persistent inflammation leads to
296
proliferative neoplasia and loss of smooth muscle cells in a prostate tumor model. Neoplasia 2011;13:692-
297
703.
13
Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2016. © 2013 American Association for
Cancer Research.
Author Manuscript Published OnlineFirst on April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
298
13. Tong S, Gwo-Shu ML, William KO, Matthew LF, Mark P, Kenneth JP, et al. Inherited variants in the
299
chemokine CCL2 gene and prostate cancer aggressiveness in a caucasian cohort. Clin Cancer Res
300
2011;17:1546-52.
301
14. Jian Z, Yi L, Kenneth JP. Multiple roles of chemokine (C-C Motif) ligand 2 in promoting prostate
302
cancer growth. J Natl Cancer Inst 2010;102:522-28.
303
15. Jian Z, Lalit P, Kenneth JP. CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis
304
and metastasis. Cytokine Growth Factor Rev 2010;21:41-8.
305
16. Loberg RD, Day LL, Harwood J, Chi Y, Lauren NJ, Ryan G, et al. CCL2 is a potent regulator of
306
prostate cancer cell migration and proliferation. Neoplasia 2006;8:578-86.
307
17. Lu Y, Cai Z, Galson DL, Galson DL, Xiao G, Liu Y, George DE, et al. Monocyte chemotactic protein-
308
1 (MCP-1) acts as a paracrine and autocrine factor for prostate cancer growth and invasion. Prostate.
309
2006;66:1311-18.
310
18. Kenneth LG, Chi Y, Linda S, C WD, Tracy R, Arul MC, et al. CCL2 induces prostate cancer
311
transendothelial cell migration via activation of the small GTPase Rac. J Cell Biochem 2008;104:1587-97.
312
19.Yanjia H, Xinchun J, Jieying P, Xiaowen J, Ning L, Shanghui Z. Gene expression profiling of oral
313
submucous fibrosis using oligonucleotide microarray. Oncol Rep 2008;20:287-94.
314
20. Bruckheimer EM, Kyprianou N. Dihydrotestosterone enhances transforming growth factor-β-induced
315
apoptosis in hormone-sensitive prostate cancer cells. Endocrinol 2001;142:2419-26.
316
21. Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, et al. Differential expression of the
317
epithelial–mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol
318
2002;161:1881-91.
319
22. Watanabe O, Imamura H, Shimizu T, Kinoshita J, Okabe T, Hirano A, et al. Expression of twist and
320
wnt in human breast cancer. Anticancer Res 2004;24:3851-56.
321
23. Kwok WK, Ling MT, Lee TW, Lau TC, Zhou C, Zhang X, et al. Up-regulation of TWIST in prostate
322
cancer and its implication as a therapeutic target. Cancer Res 2005;65:5153-62.
14
Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2016. © 2013 American Association for
Cancer Research.
Author Manuscript Published OnlineFirst on April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
323
24. Martin TA, Goyal A, Watkins G, Jiang WG. Expression of the transcription factors snail, slug, and
324
twist and their clinical significance in human breast cancer. Ann Surg Oncol 2005;12:488-96.
325
25. Amanda TP, Jurga L, Niklas M, Erik N, Anne B, Ingrid D, et al. A possible inflammatory role of
326
TWIST1 in human white adipocytes. Diabetes 2010;59:564-71.
327
26. Niesner U, Albrecht I, Janke M, Doebis C, Loddenkemper C, Lexberg MH, et al. Autoregulation of
328
Th1-mediated inflammation by twist1. J Exp Med 2008;205:1889-1901.
329
27. Sharif MN, Sosic D, Rothlin CV, Kelly E, Lemke G, Olson EN, et al. Twist mediates suppression of
330
inflammation by type I IFNs and Axl. J Exp Med 2006;203:1891-1901.
331
28. Pettersson AT, Mejhert N, Jernås M, Carlsson LM, Dahlman I, Laurencikiene J, et al. Twist1 in human
332
white adipose tissue and obesity. J Clin Endocrinol Metab. 2011;96:133-41.
333
29. Verhoeven DT, Goldbohm RA, van Poppel G, Verhagen H, van den Brandt PA. Epidemiological
334
studies on brassica vegetables and cancer risk. Cancer Epidemiol Biomarkers Prev 1996;5:733-48.
335
30. Hong C, Kim HA, Firestone GL, Bjeldanes LF. 3,3'-Diindolylmethane (DIM) induces a G(1) cell
336
cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of
337
p21(WAF1/CIP1) expression. Carcinogenesis 2002;23:1297-1305.
338
31. Chen I, McDougal A, Wang F and Safe S. Aryl hydrocarbon receptor-mediated antiestrogenic and
339
antitumorigenic activity of diindolylmethane. Carcinogenesis 1998;19:1631-39.
340
32. Daly C, Rollins BJ. Monocyte chemoattractant protein-1 (CCL2) in inflammatory disease and adaptive
341
immunity: Therapeutic opportunities and controversies. Microcirculation 2003;10:247-57.
342
33. Wang TT, Schoene NW, Milner JA, Kim YS. Broccoli-derived phytochemicals indole-3-carbinol and
343
3,3′-diindolylmethane exerts concentration-dependent pleiotropic effects on prostate cancer cells:
344
Comparison with other cancer preventive phytochemicals. Mol Carcinog 2011 Apr Epub ahead of print
345
34. Rogan EG. The natural chemopreventive vompound Indole-3-carbinol: state of the science. In Vivo
346
2006;20:221-28.
347
35. Sarkar FH, Yiwei L, Zhiwei W, Dejuan K. The role of nutraceuticals in the regulation of Wnt and
348
Hedgehog signaling in cancer. Cancer Metastasis Rev 2010;29:383-94.
15
Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2016. © 2013 American Association for
Cancer Research.
Author Manuscript Published OnlineFirst on April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
349
36. Kim YH, Kwon HS, Kim DH, Shin EK, Kang YH, Park JH, et al. 3,3'-diindolylmethane attenuates
350
colonic inflammation and tumorigenesis in mice. Inflamm Bowel Dis 2009;15:1164-73.
351
37. Oppenheim JJ, Zachariae COC, Mukaida N, Matsushima K. Properties of the novel proinflammatory
352
supergene “intercrine” cytokine family. Annu Rev Immunol. 1990;9:617-48.
353
38. Matsushima K, Baldwin ET, Mukaida N. Interleukin-8 and MCAF: Novel leukocyte recruitment and
354
activating cytokines. Chem Immunol 1992;51:236-65.
355
39. Tosato G, Jones KD. Monocytes Interleukin-1 induces interleukin-6 production in peripheral blood.
356
Blood 1990;75:1305-10.
357
40. Schetter AJ, Heegaard NHH, Harris CC. Inflammation and cancer: interweaving microRNA, free
358
radical, cytokine and p53 pathways. Carconogenesis 2010;31:37-49.
359
41. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war
360
on cancer. Oncogene Oncogene. 2010;29:4741-51.
361
42. Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med 2009;15:1010-12.
362
43. Klarmann GJ, Hurt EM, Mathews LA, Zhang X, Duhagon MA, Mistree T, et al. Invasive prostate
363
cancer cells are tumor initiating cells that have a stem cell-like genomic signature. Clin Exp Metastasis
364
2009;26:433-46.
365
44. Yuen HF, Kwok WK, Chan KK, Chua CW, Chan YP, Chu YY, et al. TWIST modulates prostate
366
cancer cell-mediated bone cell activity and is upregulated by osteogenic induction. Carcinogenesis
367
2008;29:1509-18.
368
45. Kwok WK, Ling MT, Lee TW, Lau CM, Zhou C, Zhang X, et al. Up-regulation of TWIST in prostate
369
cancer and its implication as a therapeutic target. Cancer Res 2005;65:5153-62.
370
46. Olson EN, Klein WH. bHLH factors in muscle development: dead lines and commitments, what to
371
leave in and what to leave out. Genes Dev 1994;8:1-8.
372
47. Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, et al. Twist is a
373
potential oncogene that inhibits apoptosis. Genes Dev 1999;13:2207–17.
16
Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2016. © 2013 American Association for
Cancer Research.
Author Manuscript Published OnlineFirst on April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
374
48. Yuen HF, Chua CW, Chan YP, Wong YC, Wang X, Chan KW. Significance of TWIST and E-
375
cadherin expression in the metastatic progression of prostatic cancer. Histopathology. 2007;50:648-58.
376
49. Matsushima K, Larsen CG, Dubois GC, Oppenheim JJ Purification and characterization of a novel
377
monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. J Exp Med
378
1989;169:1485-90.
379
50. Brzezińska-Błaszczyk E, Pietrzak A, Misiak-Tłoczek AH. Tumor necrosis factor (TNF) is a potent rat
380
mast cell chemoattractant. J Interferon Cytokine Res 2007;27:911-19.
381
382
17
Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2016. © 2013 American Association for
Cancer Research.
Author Manuscript Published OnlineFirst on April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
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Figure legends
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Figure 1. Effects of DHT on TWIST-1 expression in androgen-responsive LNCaP cells. LNCaP cells
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(0.25×106 cells/well) were plated in six-well plates, after androgen deprivation, the cells were daily treated
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with vehicle (DMSO) or DHT, and then subjected to RNA isolation and real-time PCR analysis. Real-time
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PCR results are expressed as means ± SE of fold change relative to vehicle-treated control of three
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separate experiments with each experiment including triplicate PCR tubes. Error bars with an asterisk are
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significantly different than vehicle-treated control (*P < 0.05, **P < 0.01, ***P < 0.001). A.
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Concentration dependent effects of DHT on TWIST-1 mRNA levels. The cells were treated daily with or
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without varied concentrations (0-1.0 nM) of DHT for 48 h. B. Time course of DHT effects on TWIST1
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mRNA levels. LNCaP cells were daily treated with or without 1 nM DHT for 0, 24, 48, or 72 h. C, D.
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Effects of AR siRNA on TWIST-1 and PSA mRNA levels. LNCaP cells were transfected with 5 nM AR
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siRNA or Negative control siRNA as described in the Materials and Methods. After transfection, the cells
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were treated with or without DHT (1nM) for 48 h as described in Materials and Methods. After treatment,
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total RNA was isolated and mRNA for PSA and TWIST-1 determined as described in Materials and
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Methods. Real-time PCR results are expressed as means ± SE of fold change relative to vehicle-treated
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control of three separate experiments with each experiment including triplicate PCR tubes. Error bars with
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an asterisk are significantly different than vehicle-treated control (*P < 0.05, **P < 0.01, ***P < 0.001). C.
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Effect of AR siRNA on DHT induction of TWIST1 mRNA levels. D. Effect of AR siRNA on DHT
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induction of PSA mRNA levels. NC: negative control siRNA.
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Figure 2. Effects of DHT on TWIST1-related genes expression in androgen-responsive LNCaP cells.
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LNCaP cells (0.25×106 cells/well) were plated in six-well plates, after androgen deprivation, the cells were
405
daily treated with vehicle or DHT for 48 h, and then subjected to RNA isolation and real-time PCR
406
analysis. Real-time PCR results are expressed as means ± SE of fold change relative to vehicle-treated
407
control of three separate experiments with each experiment including triplicate PCR tubes. Error bars with
408
an asterisk are significantly different than vehicle-treated control (*P < 0.05, **P < 0.01, ***P < 0.001).
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A. Concentration dependent effects of DHT on CDH1 mRNA levels. B. Concentration dependent effects
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of DHT on SNAIL mRNA levels. C. Concentration dependent effects of DHT on vimentin mRNA levels.
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D. Concentration dependent effects of DHT on CCL2 mRNA levels. E. Concentration dependent effects
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of DHT on TNF-α mRNA levels. F. Time course of DHT effects on CCL2 mRNA levels. G. Time course
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of DHT effects on TNF-α mRNA levels.
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Figure 3. Effects of siRNA against AR and TWIST-1 on DHT-induction of CCL2 and TNF-α mRNA
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expression in LNCaP cells. LNCaP cells were transfected with 5 nM AR siRNA, TWIST-1 siRNA or
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Negative control siRNA as described in the Materials and Methods. After transfection, the cells were
418
treated with or without DHT (1nM) for 48 h as described in Materials and Methods. After treatment, total
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RNA was isolated and mRNA for AR, TWIST-1, CCL-2 and TNF- α determined as described in Materials
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and Methods. Real-time PCR results are expressed as means ± SE of fold change relative to vehicle-
421
treated control of three separate experiments with each experiment including triplicate PCR tubes. Error
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bars with an asterisk are significantly different than vehicle-treated control (*P < 0.05, **P < 0.01, ***P <
423
0.001). A. Effects of AR siRNA on DHT-induction of CCL2 mRNAexpression. B. Effects of AR siRNA
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on DHT-induction of TNF-α mRNA expression. C. Effects of TWIST-1 siRNA on DHT-induction of
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CCL2 mRNA expression. D. Effects of TWIST-1 siRNA on DHT-induction of TNF-α mRNA expression.
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E. Effects of AR or TWIST-1 siRNA on AR (left panel) and TWIST-1 (right Panel) mRNA expression.
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NC: negative control siRNA.
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Figure 4. Baseline and DHT-induced mRNA levels of TWIST-1, CCL-2 and TNF- α in androgen-
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responsive LNCaP and non-responsive PC-3 cells. LNCaP or PC-3 cells were plated in six-well plates,
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after androgen deprivation, the cells were daily treated with vehicle or DHT for 48 h, and then subjected to
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RNA isolation and real-time PCR analysis. Real-time PCR results are expressed as means ± SE of fold
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change relative to vehicle-treated control of three separate experiments with each experiment including
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triplicate PCR tubes. Error bars with an asterisk are significantly different than vehicle-treated control
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(***P < 0.001). A. Relative baseline mRNA levels of TWIST-1, CCL-2 and TNF- α in LNCaP and PC-3
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cells. B. Effect of DHT on genes expression in androgen-responsive LNCaP cells. C. Effects of DHT on
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genes expression in androgen-nonresponsive PC-3 cells.
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Figure 5. Effect of DHT on CCL2 protein and LNCaP cell conditioned media-stimulated THP-1 monocyte
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migration. LNCaP cells were treated with or without 1 nM DHT for 48 h, conditioned media harvested
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and CCL2 protein level, THP-1 migration assay performed as described in Materials and Methods. The
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results are expressed as means ± SE of fold change relative to vehicle-treated control of three separate
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experiments. Error bars with an asterisk are significantly different than vehicle-treated control (**P <0.01,
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***P < 0.001. as analyzed by Bonferroni post-test). A. Protein expressions of CCL2 in LNCaP cells
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conditioned media. Error bars with an asterisk are significantly different than vehicle-treated control (**P
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<0.01, ***P < 0.001). B. Effect of conditioned media on THP-1 cells migration. Error bars with an
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asterisk are significantly different than vehicle-treated control (**P <0.01, ***P < 0.001). C. Effect of
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CCL2 Blocking antibody on conditioned media-induced THP-1 migration. LNCaP cells conditioned
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media were treated with 0.5 μg/ml isotype control or CCL2 blocking antibody for 1 h then the THP-1
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migration assay was conducted as described in Materials and Methods. Values with a different superscript
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are significantly different at p<0.05.
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Figure 6. Effect of I3C and DIM on DHT-induced gene expression in LNCaP cells and conditioned media
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stimulated THP-1 cell migration. LNCaP cells were treated with or without 1 nM DHT for 48 h, cell
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harvested for total RNA isolation and gene expression analysis as described in Materials and Methods.
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Conditioned media were harvested for CCL2 protein ELISA and THP-1 migration assay as described in
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Materials and Methods. The results are expressed as means ± SE of fold change relative to vehicle-treated
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control of three separate experiments. A. I3C and DIM inhibition of DHT-induced PSA expression. B. I3C
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and DIM inhibition of DHT-induced TWIST-1 mRNA expression. C. I3C and DIM inhibition of DHT-
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induced CCL2 mRNA expression. D. I3C repressed the protein expression of CCL2 in DHT-treated
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LNCaP media E. DIM repressed the protein expression of CCL2 in DHT-treated LNCaP media. F. I3C
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suppressed THP-1 migration in DHT-treated LNCaP conditioned media. G. DIM suppressed THP-1
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migration in DHT-treated LNCaP conditioned media. Error bars with an asterisk are significantly different
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than vehicle-treated control (*P <0.05, **P <0.01, ***P < 0.001).
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Indole-3-carbinol and 3', 3'-diindolylmethane modulate
androgen's effect on C-C chemokine ligand 2 and monocyte
attraction to prostate cancer cells
Eun-Kyung Kim, Young S. Kim, John A. Milner, et al.
Cancer Prev Res Published OnlineFirst April 12, 2013.
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